A Novel Fibrinogen Assay Using Recombinant Batroxobin and Carboxymethyl Chitosan: Carboxymethyl Chitosan Stimulates the Enzymatic Activity of Recombinant Batroxobin
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Background: The Clauss assay is widely used to quantify blood fibrinogen levels in clinical laboratories. However, by relying on thrombin as the main reagent, the Clauss assay is sus- ceptible to interference from thrombin inhibitors, such as heparin or direct thrombin inhibi- tors. Here, we developed an innovative fibrinogen assay utilizing both recombinant ba- troxobin (rBat) and carboxymethyl chitosan (CMCS). Methods: Various biopolymers were tested to identify a suitable candidate that could en- hance rBat-induced fibrin clot formation. Chromogenic substrate hydrolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that CMCS potentiated rBat activity. Consequently, we formulated a novel fibrinogen assay reagent, ANYFIB.C, comprising rBat and CMCS. We compared ANYFIB.C fibrinogen with an established re- agent (HemosIL fibrinogen-C) with 96 clinical samples using an ACL-TOP 700 coagulation analyzer. We also evaluated the interfering effects of thrombin inhibitors on fibrinogen measurements. Results: CMCS significantly enhanced the enzymatic activity of rBat and dose-dependently reduced plasma clotting times. ANYFIB.C fibrinogen levels were comparable with those of HemosIL fibrinogen-C, with the 95% confidence intervals of the Passing–Bablok regres- sion intercept and slope being −7.4797 to 6.0185 and 0.9581 to 1.0116, respectively. No significant interference was observed with heparin concentrations up to 10 U/mL or dabi- gatran concentrations up to 600 µg/L in the ANYFIB.C fibrinogen assays. In contrast, the HemosIL fibrinogen-C reagent demonstrated inhibitory interference at dabigatran concen- trations as low as 150 µg/L. Conclusions: Our results suggest that ANYFIB.C (a mixture of CMCS and rBat) can be used to measure blood fibrinogen levels effectively and protect from thrombin inhibitor interfer- ence.
Abstract
Background: The Clauss assay is widely used to quantify blood fibrinogen levels in clinical laboratories. However, by relying on thrombin as the main reagent, the Clauss assay is sus- ceptible to interference from thrombin inhibitors, such as heparin or direct thrombin inhibi- tors. Here, we developed an innovative fibrinogen assay utilizing both recombinant ba- troxobin (rBat) and carboxymethyl chitosan (CMCS). Methods: Various biopolymers were tested to identify a suitable candidate that could en- hance rBat-induced fibrin clot formation. Chromogenic substrate hydrolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that CMCS potentiated rBat activity. Consequently, we formulated a novel fibrinogen assay reagent, ANYFIB.C, comprising rBat and CMCS. We compared ANYFIB.C fibrinogen with an established re- agent (HemosIL fibrinogen-C) with 96 clinical samples using an ACL-TOP 700 coagulation analyzer. We also evaluated the interfering effects of thrombin inhibitors on fibrinogen measurements. Results: CMCS significantly enhanced the enzymatic activity of rBat and dose-dependently reduced plasma clotting times. ANYFIB.C fibrinogen levels were comparable with those of HemosIL fibrinogen-C, with the 95% confidence intervals of the Passing–Bablok regres- sion intercept and slope being −7.4797 to 6.0185 and 0.9581 to 1.0116, respectively. No significant interference was observed with heparin concentrations up to 10 U/mL or dabi- gatran concentrations up to 600 µg/L in the ANYFIB.C fibrinogen assays. In contrast, the HemosIL fibrinogen-C reagent demonstrated inhibitory interference at dabigatran concen- trations as low as 150 µg/L. Conclusions: Our results suggest that ANYFIB.C (a mixture of CMCS and rBat) can be used to measure blood fibrinogen levels effectively and protect from thrombin inhibitor interfer- ence.